Preparation of media & sterilization technique

culture medium and their types,
procedure for both nutrient and agar &
sterilization methods & techniques

1. - Fathima Hameed



Contents
 Media
 Types of media
 Materials required
 Preparation of media
 Sterilization technique
 Sterilization methods
 References

2. Media
 Bacteria and fungi are grown on or in microbiological media of various types.
 Different nutrients may be added to the medium, making it higher in protein or in sugar. Various pH
indicators are often added.
 In this exercise, you will make all-purpose media called nutrient broth (liquid) and nutrient agar (solid)
are the exact same formula save for the addition of agar agar, an extract from the cell walls of red algae.
 The most common growth media nutrient broths or LB medium are liquid.
 These are often mixed with agar and poured into Petri dishes to solidify.
 These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as
very few bacteria are able to decompose agar. Many microbes can also be grown in liquid cultures
comprised of liquid nutrient media without agar.

3. Types of media
 A defined medium will have known quantities of all ingredients like, trace elements, vitamins carbon
and nitrogen source. Glucose or glycerols are often used as carbon sources, and ammonium salts or
nitrates as inorganic nitrogen sources.
 An undefined medium has some complex ingredients, such as yeast extract, which consists of a mixture
of many, many chemical species in unknown proportions.

Based on ingredients, medium classified as various types
1) Nutrient media – A source of amino acids and nitrogen (e.g., beef, yeast extract). This is an undefined
medium because the amino acid source contains a variety of compounds with the exact composition
being unknown. These media contain all the elements that most bacteria need for growth and are non-
selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory-
culture collections.

2) Minimal media – Media that contains the minimum nutrients possible for colony growth, generally
without the presence of amino acids, and are often used by microbiologists and geneticists to grow “wild
type” microorganisms. These media can also be used to select for or against the growth of specific
microbes. Usually a fair amount of information must be known about the microbe to determine its
minimal media requirements.


3) Selective media – Used for the growth of only selected microorganisms. For example, if a
microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic
can be added to the medium in order to prevent other cells, which do not possess the resistance, from
growing.

4) Differential media – Also known as indicator media are used to distinguish one microorganism type
from another growing on the same media. This type of media uses the biochemical characteristics of a
microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol
red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a
microorganism. This type of media is used for the detection and identification of microorganisms.

4. Based on physical state, medium classified as 3 types
1. Solid medium – contains solidifying agent (agar)
2. Liquid medium – without agar (broth) & producing turbidity (growth)
3. Semi solid medium – demonstrating bacterial motility



Materials required
 Nutrient broth & agar
 Agar agar
 Distilled water
 Conical flask
 Beaker
 Culture tubes
 Cotton
 Glass L rod
 Petri plates

5. Preparation of media
Procedure for nutrient broth
1. Put the few amount of nutrient broth in 100 ml distilled water.
2. Adjust pᴴ of the medium to 7 using a pᴴ meter, while adding either acid or alkali, as the case may
be.
3. Pour 10 ml of broth in each test tube.
4. Apply cotton plug.
5. Autoclave at 121º C, 15 lbs pressure for 15 minutes.
6. After sterilization, the nutrient broth tubes stored at refrigerator for further use.

Procedure for nutrient agar
1. Weighed a few amount of nutrient agar medium & agar agar dissolved in 100 ml of distilled
water.
2. Allow it cool & 20 ml of agar medium was poured into each petriplates and allowed to solidify.
3. Take a 0.1 ml of bacterial culture poured on the surface of the medium.
4. The bacterial suspension was spread by L rod.
5. Then all plates were incubated at 37 º C for 24-48 hrs.
Sterilization technique
 When microbiological media has been made, it still has to be sterilized because of microbial
contamination from air, glassware, hands, etc. Within a few hours there will be thousands of bacteria
reproducing in the media so it has to be sterilized quickly before the microbes start using the nutrients
up.
 The sterilization process is a 100% kill, and guarantees that the medium will stay sterile. Media
sterilization is carried out with the autoclave, basically a huge steam cooker. Steam enters into a jacket
surrounding the chamber. When the pressure from the steam is at a certain point in the jacket, a valve
allows the steam to enter the chamber.
 The pressure will go up over 15 pounds per square inch (psi): at this point the timer begins to count
down--- usually for 15 minutes, depending on the type of media. The high pressure in a closed container
allows the temperature to go above the highest temperature one could get by just boiling, around 121⁰C.
Therefore, the parameters for sterilization with an autoclave are 121⁰C at >15 psi for 15 minutes.

6. Methods of sterilization
Physical methods
1. Moist heat – autoclave & pressure cooker
2. Dry heat – hot air oven
3. Radiation – UV radiation
4. Sterilization by membrane filtration – Millipore membrane filter
Chemical methods
1. Alcohol – ethanol & isopropanal (70%)
2. Aldehyde – formaldehyde
3. Inorganic chemicals – heavy metals; copper, mercury, etc.


References
• https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiology_Labs
/Microbiology_Labs_I/01%3A_Media_Preparation
• https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/6%3A_Cult
uring_Microorganisms/6.3%3A_Culturing_Bacteria/6.3A%3A_Culture_Media#:~:text=Culture%20med
ium%20or%20growth%20medium,such%20as%20bacteria%20or%20yeast.
• https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.slideshare.net%2FHiwrHastear%2Ftyp
es-of-culture-
media&psig=AOvVaw1SW3wSCoXlEnLGy2woeFZt&ust=1602393645949000&source=images&cd=v
fe&ved=0CAMQjB1qFwoTCICQm9qjqewCFQAAAAAdAAAAABAQ